Comb copolymers for regulating cell-surface interactions

ABSTRACT

Synthetic comb copolymers which elicit controlled cellular response, methods of applying these polymers to various surfaces, and methods of using the polymers for modifying biomaterial surfaces, in tissue engineering applications and as drug delivery devices are provided. The comb copolymers are comprised of hydrophobic polymer backbones and hydrophilic, non-cell binding side chains which can be end-capped with cell-signaling ligands that guide cellular response. By mixing non-cell binding combs with ligand-bearing combs, the surface concentration and spatial distribution of one or more types of ligands, including adhesion peptides and growth factors, can be tuned on a surface to achieve desired cellular response. In one embodiment, the combs are used as stabilizing agents for dispersion polymerization of latexes. The comb-stabilized latexes can be applied to substrates by standard coating operations to create a bioregulating surface, or used as drug delivery agents. In another embodiment, the combs can be blended in small quantities to a hydrophobic matrix polymer and processed to affect the surface segregation of the comb. The comb copolymers are formed in one embodiment by providing a biodegradable polyester backbone that includes reactive groups, and reacting the reactive groups in the backbone with reactive chain ends on a low molecular weight hydrophilic polymer. In another embodiment, non-biodegradable comb copolymers are formed by free radical synthesis of a hydrophobic monomer and a hydrophilic macromonomer. In all of the above embodiments, a portion of the hydrophilic polymer side chains can be covalently coupled to cell-signaling ligands such as adhesion peptides or growth factors to control cellular response.

This application is a continuation of U.S. Ser. No. 09/634,095 filedAug. 8, 2000 now U.S. Pat. No. 6,207,749, which is a divisional of U.S.Ser. No. 09/290,140 filed Apr. 13, 1999, now U.S. Pat. No. 6,150,459which issued Nov. 21, 2000, which priority to U.S. Ser. No. 60/081,596filed Apr. 13, 1998.

The United States government has certain rights in this invention byvirtue of National Science Foundation grant No. DMR-9400334, OSP ProjectNo. 6227 to Anne M. Mayes, and National Science Foundation Grant No. BES9632714 to L. G. Griffith.

BACKGROUND OF THE INVENTION

Polymeric materials that elicit controlled cell responses, and have goodmechanical, optical and/or biodegradation properties, are disclosed foruse in biomedical applications. Processing methods by which suchpolymers can be localized at a biomaterial surface are also disclosed.

Polymers currently in use for biomedical applications generally tend tobe hydrophobic. As defined herein, hydrophobic refers to a material thatrepels water, i.e., exhibits a static contact angle with water greaterthan 60 degrees at 20° C., and has a water permeability P less than3×10⁻¹⁰ cm³(STP) cm/(cm² s Pa). This can give rise to uncontrolledinteractions between cells and adsorbed proteins at the surface of thematerial, which can result in a chronic inflammatory response that canlead to failure of implants and even promote tumorigenecity (Warson, TheApplications of Synthetic Resin Emulsions, Benn, London (1972)). Metalor ceramic materials used in implant applications similarly can elicitundesirable cell responses.

For tissue engineering applications, it is essential that the polymericmaterial used to form a biodegradable scaffold for cells promote celladhesion, migration, growth and differentiation while providing adequatestructural support. Though commonly used synthetic scaffold materialssuch as poly(lactide), poly(glycolide), etc., and copolymers thereof,have suitable mechanical, processing and biodegradation properties,their hydrophobic nature leads to protein adsorption and denaturing onthe material surface which elicits uncontrolled cell response.

The ideal surface for many biomaterials applications would resistprotein adsorption while providing cells with specific chemical signalsto guide adhesion, survival, growth, migration and differentiation. Asused herein, the term “biomaterial” refers to a nonviable material usedin a medical device intended to interact with biological systems.Polymer surfaces modified with poly(ethylene oxide) have been studied inrecent years for the reduction of protein adsorption at the surface ofbiomaterials (Paine et al. Macromolecules, 23:3104 (1990)). Theobjective of these surface modification schemes is the elimination ofnonspecific interactions of cells with implant materials. One way inwhich specific chemical signals can be relayed to cells at a surface isthrough tethered ligands for cell surface receptors (Barret, Brit.Polym. J. 5:259 (1973)). Delivery of signals in this manner hasadvantages over the addition of soluble factors, as the signal ispresented in a very localized manner at a controlled dose withoutdiffusive loss (Kuhl and Griffith, Nature Medicine, 2:1002 (1996)). Inaddition, tethered ligands may provide more constant stimulation tocells by avoiding the down-regulation present when soluble ligands areinternalized by cells. Control over spatial distribution of ligands onsurfaces may also be key to guiding cell behavior. Thus systems whichwill allow spatial control of local ligand density, or the creation ofclusters of ligands on a surface, in addition to providing control overthe average surface density of ligands, are highly desirable (Kornberget al, Proc. Natl. Acad. Sci. USA, 88:8392 (1991)).

Integrins, dimeric adhesion receptors including one of approximately tenknown alpha chains paired with one of approximately six known betachains, mediate a wide range of interactions between cells andextracellular matrix (ECM) and control cell behaviors as diverse asmigration, growth, and differentiation, providing a permissiveenvironment for the action of growth factors. For many integrins, thespecificity of integrin binding to matrix proteins has been mapped tosmall, discrete peptide domains and new sites continue to be elucidated(Rouslahti, Ann. Rev. Cell. Dev. Biol., 12:697 (1996); Hynes, Cell,48:549 (1987)). The prototypical example of such specificity is the RGDsite first identified in fibronectin and subsequently identified inother matrix proteins. The RGD peptide enables complete replacement ofadhesive finction of fibronectin for cells expressing certain integrins.

Much data supports the idea that both occupancy and clustering ofintegrins are required to elicit full cellular responses mediated byintegrins (Clark and Brugge, Science, 268:233 (1995)). For example, fullEGFR activation of MAP kinase requires integrin clustering and occupancy(Miyamoto et al, J. Cell Biol., 135:1633 (1996)). Thus, the spatialpresentation of ligand in the environment, i.e., whether ligands arespaced closely enough to afford clustering of ligand-bound integrins,may influence cellular behaviors governed by integrins. Indeed, spacingof synthetic RGD ligand covalently linked to the substrate has beenshown to have an influence on cell adhesion and spreading (Massia andHubbell, J. Cell Biol., 114:1089 (1991)). At the same time, the surfaceconcentration of an adhesion ligand such as fibronectin has been shownto have a substantial influence on integrin-mediated behaviors such asmigration (DiMilla et al, J. Cell Biol., 122:729 (1993)). A recent studyusing self-assembled monolayers patterned in one micronadhesive/nonadhesive domains demonstrated the role of cell spreading andreceptor occupancy on cell survival (Chen et al, Science, 276:1425(1997). The length scale in that study was approximately that of a focaladhesion complex (or larger), but it is likely that clustering over muchsmaller length scales (3-10 integrins) is also physiologically relevant.Indeed, data suggests strongly that RGD clustering on the less than 100nm length scale has profound effects on the integrin-mediated behaviorof migration. Since both the concentration and spatial distribution ofligand influence cell response, it is desirable to have a means to varythese two parameters independently, and over a broad range of lengthscales (nanometers to micrometers), in order to guide cell response.

Integrins can initiate intracellular signaling cascades that overlapwith those of growth factors such as epidermal growth factor (EGF).Cross-communication between adhesion and growth factor receptors mayoccur by direct physical association within the focal adhesions. Bothtypes of receptors are concentrated in these structures (Miyarnoto etal, J. Cell Biol., 135:1633 (1996); Plopper et al, Mol. Biol. Cell,6:1349 (1995)), and both receptors can stimulate some of the samedown-stream effect on molecules such as MAP kinase. Close proximity ofadhesion and growth factor receptors in the focal adhesion complexprovides for a free flow of both positive and negative regulatorysignals between the two. A number of signaling molecules have beenproposed as forming this linkage; one intracellular mechanism oftransmodulation is via protein kinase C (PKC)-mediated attenuation ofthe epidermal growth factor receptor (EGFR). It is also likely that PKCactivity secondary to phospholipase Cγ or phospholipase D activation byEGFR alters integrin-based substratum connections (Welsh et al, J. CellBiol., 114:533 (1991); Ando et al, J. Cell. Physiol., 156:487 (1993)).It is thus desirable to have a method by which two or more types ofsignaling ligands, such as adhesion peptides and growth factors, can besimultaneously located at the surface of a biomaterial in controlledquanitity and spatial distribution.

To date, few if any model systems are able to meet both proteinresistance and cell signaling surface requirements, while approachesusing clinically-applicable materials have focused on hydrogels (Hernand Hubbell, J. Biomed. Mater. Res., 39:266 (1998)), which have limitedphysical strength and are not suitable for many applications. Otherapproaches for modifying the surfaces of hydrophobic polymeric materialsor other biomaterials to achieve a more desirable surface compositionfor biomedical applications include adsorption of block copolymers,chemical grafting of polymers to the surface, and plasma deposition ofan overlying film. Each of these methods suffers various disadvantages.For example, adsorbed block copolymers can be rearranged actively bycells, grafted polymers are difficult to apply at high density on asurface, and plasma deposition results in a gel-like surface structurepoorly suited for controlled cell signaling. None of these methodsprovides a means for modifying the surface of complex three-dimensionalstructures such as fibrous or sponge-like tissue scaffolds, or forcreating clustered ligand distributions of variable concentration andspacing on biomaterial surfaces.

It would be advantageous to provide polymer materials and processingmethods that overcome the disadvantages of other biomaterials surfacemodification approaches. It is therefore an object of the presentinvention to provide polymer materials that elicit controlledcell-surface interactions by inhibiting protein adsorption, and, whereappropriate, presenting controlled concentrations and spatialdistributions of cell-signaling ligands on biomaterial surfaces. It isfurther an object of the present invention to provide processing methodsby which such polymers can be placed at a biomaterial surface. It isfurther the object of the present invention to provide polymericmaterials which can be used to create discrete nanometer- tomicrometer-sized domains on a biomaterial surface that present two ormore different types of ligands for regulating cellular response.

SUMMARY OF THE INVENTION

Comb-type copolymers that elicit controlled cellular response, methodsby which such polymers can be localized at a surface, and methods ofusing such polymers for modifying the surfaces of biomedical devices aredisclosed.

The polymers include a hydrophobic, water-insoluble backbone and lowmolecular weight, hydrophilic, non-cell binding side chains. As definedherein, non-cell binding refers to materials which exhibit no observablecell attachment after standard cell culturing assays in serum containingmedia for 24 hours. The molecular weight of the hydrophilic side chainsis preferably above 200 Daltons and below 2000 Daltons. The backbone canbe biodegradable or non-biodegradable, depending on the intendedapplication. Biodegradable backbones are preferred for most tissueengineering, drug delivery and wound healing device applications, whilenon-biodegradable backbones are desirable for permanent implant,biofiltration, and cell culture plate applications A portion of thenon-cell binding side chains can be end-capped with cell-signalingligands to control the degree of cell adhesion, or other cell response,elicited by the polymer surface. In the preferred embodiment, theoverall comb copolymer should have a molecular weight sufficiently highas to confer good mechanical properties to the polymer in the melt statethrough chain entanglements. That is, its molecular weight should beabove the entanglement molecular weight, as defined by one of ordinaryskill in the art. The overall molecular weight of the comb copolymershould thus be above about 10,000 Daltons, more preferably above 20,000Daltons, and more preferably still above 30,000 Daltons.

The density of the hydrophilic side chains along the backbone of thenoncopolymers depends on the length of the side chains and thewater-solubility characteristics of the final polymer. The totalpercentage by weight of the hydrophilic side chains is between 20 and 60percent of the total copolymer composition, preferably around 40 percentby weight. For combs incorporating hydrophilic side chains with amolecular weight of about 350 Daltons, the mole percent of segments ofthe backbone carrying hydrophilic side chains can be as high as 30percent. For hydrophilic side chains with a molecular weight of about2000 Daltons, the mole percent of segments of the backbone carryinghydrophobic side chains can be as low as 2 percent. In the preferredembodiment, the overall comb copolymer is not water-soluble. As definedherein, the term water-soluble refers to materials having a solubilityin aqueous solutions of greater than 1 gram per liter. When in contactwith aqueous solutions, the hydrophilic side chains swell and form ahydrated layer which repels proteins and hence resists cellularadhesion.

The non-cell binding side chains of the comb copolymer can be end-cappedwith cell-signaling chemical ligands in order to elicit controlled cellresponses Ligands such as adhesion peptides or growth factors can becovalently or ionically attached to the ends of the side chains usingknown chemistries to provide specific chemical signals to cells. Adefined fraction of ligand-bearing side chains can be obtained by usingappropriate stoichiometric control during the coupling of the ligands tothe polymers, by protecting the end-groups on those side chains whichare not to be end-capped with ligands, or by combinations of theseapproaches. For applications where it is desirable to cluster ligands onthe length scale of nanometers or tens of nanometers on a biomaterialsurface, more than one ligand (on average) can be covalently attached toa single comb copolymer chain. In applications where it is desirable toincorporate two or more types of ligands in a single cluster on abiomaterial surface on the size scale of nanometers to tens ofnanometers, one or more of each of the ligand types (for example, anadhesion peptide and growth factor) can be attached to a single combcopolymer chain through its side chains using known chemistries.

When adhesion peptides are coupled to the comb copolymer side chains,cells attach and spread readily on the comb copolymer surface. Theamount of cell spreading and proliferation on the surface therefore canbe controlled by mixing adhesion peptide-bearing comb copolymers withnon-cell binding comb copolymers, for example, so that less than 20% ofthe combs bear an adhesion peptide. Similarly, the spatial distributionof ligand clusters on the biomaterial surface can be controlled bymixing non-cell binding comb copolymers with comb copolymers in whicheach chain on average has more than one ligand attached to its sidechains In this case, the size of the ligand clusters (i.e., the spatialarea in which the ligands are localized) is dictated by thecharacteristic size of the ligand-bearing comb copolymer, and can beapproximated from the comb copolymer's radius of gyration, R_(G), whichcan be calculated or experimentally determined by one of ordinary skillin the art. The comb copolymer radius of gyration can range typicallybetween nanometers and several tens of nanometers, depending on totalmolecular weight, length of side chains, and environment surrounding thepolymer chain, for example, other polymer chains or water molecules(P.-G. deGennes, Scaling Concepts in Polymer Physics, Cornell UniversityPress, 1979). Thus the size of the ligand clusters, as well as thenumber and type of ligands per cluster, can be controlled by thesynthesis conditions of the ligand-bearing comb copolymers. For example,a comb copolymer with R_(G)==4 nm would have an area per cluster of πR_(G) ² or approximately 50 nm². The number of clusters on the surfaceper unit surface area (on average) can be controlled by the ratio ofligand-bearing to non-cell binding combs at the surface. To achieve asurface separation distance between ligand clusters of d, whered>2R_(G), the concentration of ligand-bearing combs should beapproximately φ=V_(chain)/(2R_(G)d²), where V_(chain) is the volumeoccupied by a single comb copolymer chain. For example, to achieve acluster-to-cluster distance of 20 nm with a comb copolymer which hasR_(G)=4 nm and V_(chain)=48 nm³, the estimated fraction ofligand-bearing combs required is 1.5 vol %. A cluster-to-clusterdistance of 10 nm would require 6 vol % of the ligand-bearing comb.

Numerous methods can be used to apply the comb copolymers, or theirmixtures, to various biomaterial surfaces. These methods include dipcoating, spray coating, brush coating, roll coating, or spin casting afilm onto the substrate, typically followed by mild heating to promoteadhesion to the surface. Solid free form processes such as threedimensional printing techniques (3DP), or freeze drying methods could beused to create complex three-dimensional structures, including porousstructures. In all of these processing approaches a suitablecrosslinking agent might be incorporated to enhance the mechanicalrigidity of the film or device.

In applications where it is desirable to use only small amounts ofcopolymer to modify the surface of a second, hydrophobic or non-cellregulating polymer, the comb copolymers can be added in small quantitiesto the second polymer and processed to achieve segregation of the combcopolymer to the surface. In preferred embodiments, the comb copolymerwould comprise less than 10 wt % of the polymer mixture. Processingsteps to achieve segregation include heating the mixture under a vacuum,in air, water, water vapor, CO₂ or other environment which favors thecomb component at the surface, at temperatures sufficiently above theglass transitions of the polymer components to provide mobility forachieving surface segregation. In the case where the second polymercomponent is a semicrystalline polymer, the annealing temperature shouldbe above the glass transition but below the melting point of thepolymer, to ensure that the desired shape of the device is retained. Inpreferred embodiments, surface segregation is achieved during a standardprocessing step in the manufacture of a biomedical device, such asduring an extraction, autoclaving or sterilization process. In otherembodiments, segregation is accomplished in an additional annealing stepin a controlled environment (water, etc), after device fabrication. Suchprocessing steps create a surface layer approximately 2R_(G) inthickness that contains almost exclusively the comb copolymer. Theobservable surface properties of such annealed mixtures aresubstantially identical to those of the pure comb copolymers. Inpreferred embodiments, the comb copolymer is miscible with the secondpolymer to avoid phase separation in the bulk device, which might leadto poor mechanical or optical properties.

In other cases, the localization of the comb polymer to the surface of adevice primarily comprised of a second, hydrophobic or non-cellregulating polymer can be accomplished during other steps of devicemanufacture. For example, precise placement of the comb copolymer at thesurface of a device made from a second polymer can be accomplished by3DP methods. Likewise, differences in viscosity between the combcopolymer and a second polymer when blended together can be exploited tolocate the comb to the surface during melt extrusion of fibers, films orother devices. Porous or nonporous membranes, films, fibers or hollowfibers in which the comb copolymer resides at the surfaces can beprepared by phase inversion casting. In this method, a solution of thecomb copolymer, the second polymer, and a mutual solvent is cast into anaqueous-based coagulation bath to form the device During the castingprocess, favorable interactions between the comb and the coagulationbath medium induce segregation of the comb copolymer to exteriorsurfaces of the film, fiber, or membrane. Cell-regulating microporousbiodegradable membranes useful as temporary barrier devices inwound-healing applications can be prepared in this fashionCell-regulating biodegradable sutures can similarly be prepared byspinning fibers from solution into an aqueous-based coagulation bath.Such surface-modified fibers can also be prepared from biodegradable ornonbiodegradable materials and fashioned into nonwoven fabric articlesfor biomedical applications including cell-regulating temporary barrierdevices and biofiltration devices. Hollow nanoporous fibers can beprepared which have cell-regulating interior surfaces. By encapsulatingcells in a portion of such a fiber, a long-term drug delivery implantcould be prepared which secretes desirable products of cells inquantities regulated wholly or in part by tethered signals on the fiberinner surface. Cell-regulating biodegradable microporous scaffolds witha surface excess of comb copolymers can be prepared by freeze-dryingmethods by choosing a sublimating solvent which has preferentialaffinity to the comb copolymer component as compared to the secondpolymer component which forms the bulk of the device.

In all cases described above where comb copolymers are used inconjunction with a second polymer to prepare a device, the combcopolymers can be non-cell binding combs, ligand-bearing combs, or amixture of these to achieve a desired cell response as previouslydescribed.

A further method by which the comb copolymers can be used forcontrolling cell response in biomedical applications is through thepreparation of polymer latexes that incorporate the comb copolymers onthe latex particle surfaces. Such latexes are prepared by dispersion oremulsion polymerization methods in a water-containing medium, using thecomb copolymers as a stabilizing agent. The polymerization is achievedby dissolving or mixing the desired monomer, comb stabilizer andinitiator in a water-containing medium. The polymer is initiated, forexample, by applying heat to the solvent. The dispersion medium is agood solvent for the comb copolymer but a poor solvent for the growingpolymer The hydrophobic comb backbone is chosen to be compatible withthe polymer being synthesized, and thus anchors to the surface of thegrowing polymer particles, while the hydrophilic side chains stabilizethe particles against flocculation Upon completion of the latexsynthesis, the resulting latex particles are in the range of 0.1 to 10μm in size, typically dispersed at 20-70% polymer solids by weight inthe dispersion medium. These systems can be employed in a variety ofways to control cell response through the comb copolymers that remainanchored to the particle surfaces.

Films or coatings can be prepared from the latex dispersions by usualmethods such as dipping, brushing, rolling or casting the latex onto anysurface. For coatings applied to permanent implants to control cellresponse, nonbiodegradable latex particles are preferred, such asacrylics. Opaque coatings may be prepared that elicit controlled cellresponse by employing any of the standard coating methods used to formlatex films by those skilled in the art, such as those just mentioned.Alternatively, by heat-treating films at a temperature well above theglass transition of the polymer particles, the particles will coalesceinto a smooth, transparent film in which the comb copolymers reside atthe surface. The comb copolymers remain localized at the surface uponcoalescence due either to an energetic tendency to remain at thesurface, or because there is insufficient mobility for comb diffusioninto the bulk of the coalesced latex film, for example, if the film iscooled below its glass transition shortly after coalescence. The latexfilms exhibit surface properties akin to the comb copolymers themselves,but have the advantages that only small quantities of the comb copolymerare used (typically below 1 wt % of the total latex), coatings can beeasily applied from water-based suspensions, and the film-formingproperties can be tailored to adhere to the substrate by judiciouschoice of the film-forming polymer. For example, an acrylic latexstabilized by non-cell binding comb copolymers could be used to preparetransparent acrylic coatings on acrylic intraocular lenses in order torender them resistant to cell attachment, and hence less subject toclouding over time Acrylic latexes could also be used in applicationswhere controlled cell response is desired at the surface of permanentmetal, glass or ceramic implants or other devices, including cellculture apparatus, since a high degree of adhesion is often foundbetween oxide surfaces and acrylic polymers. For polystyrene cellculture plates or other apparatus, a cell-regulating PS latex could beused to prepare a transparent, cell-regulating coating in the mannerdescribed above.

In all cases described above where latexes are stabilized by combcopolymers, the comb copolymers might be non-cell binding combs,ligand-bearing combs, or a mixture of these to achieve a desired cellresponse as previously described above. Alternatively, mixed latexdispersions can be used to prepare films which contain clustered ligandregions on a surface of sizes from 0.1 to 10 micrometers. This can beachieved by mixing together dispersions of latex particles coated withnon-cell binding combs and those coated with ligand-bearing combs andcreating films of these mixed dispersions as described above. The sizeof the ligand clusters is approximately the diameter of the latexparticles coated with ligand-bearing combs, while the number of clusterson the surface per unit surface area can be controlled by the ratio ofligand-bearing to non-cell binding latex particles in the mixeddispersion.

For applications where a biodegradable film is preferred, biodegradablelatexes can be prepared using comb stabilizers with biodegradablebackbones. Such biodegradable latexes could also be employed as drugdelivery vehicles as described below.

DETAILED DESCRIPTION OF THE INVENTION

Comb-type copolymers that elicit regulated cellular response, methods bywhich such polymers can be localized at a surface, and methods of usingsuch polymers for modifying the surfaces of biomedical devices aredisclosed.

These polymers are characterized by properties that are a function ofthe type and ratio of hydrophilic side chains to hydrophobic backbonepolymers, type and number of tethered cell-signaling ligands, molecularweight, and processing conditions

I. Polymer Composition

A. Polymer Architecture

The polymers are comb-type copolymers, with a backbone formed of ahydrophobic, water-insoluble polymer and side chains formed of short,hydrophilic non-cell binding polymers, having a molecular weight ofbetween 200 and 2000 Daltons. The hydrophobic backbone can bebiodegradable or non-biodegradable, depending on the desiredapplication. The overall comb copolymer should have a molecular weightsufficiently high in the melt state as to confer good mechanicalproperties to the polymer through chain entanglement, that is, itsmolecular weight should be above the entanglement molecular weight, asdefined by one of ordinary skill in the art. The overall molecularweight of the comb copolymer should thus be above about 10,000 Daltons,more preferably above 20,000 Daltons, and more preferably still above30,000 Daltons. The comb copolymers can be prepared by copolymerizing ahydrophilic macromonomer which contains a polymerizable chain end with asecond hydrophobic monomer. Alternatively, a hydrophobic monomer can becopolymerized with a second monomer that includes suitable reactivegroups through which the hydrophilic side chains can be grafted to thebackbone. Alternatively, a hydrophobic monomer with a suitable reactiveside group can be polymerized and a fraction of those reactive sidegroups can be modified by grafting hydrophilic side chains. A definedpercentage of the non-cell binding side chains can be end-capped with asuitable ligand to elicit a specific cellular response.

B. Hydrophobic Polymer Backbones

1. Biodegradable Hydrophobic Polymers

Hydrophobic polymers used to impart biodegradable properties to thebackbones of the comb copolymers are preferably hydrolyzable under invivo conditions. Suitable biodegradable polymeric units include hydroxyacids or other biologically degradable polymers that yield degradationproducts that are non-toxic or present as normal metabolites in thebody. These include poly(amino acids), poly(anhydrides),poly(orthoesters), and poly(phosphoesters). Polylactones such aspoly(epsilon-caprolactone), poly(delta-valerolactone),poly(gamma-butyrolactone)and poly(beta-hydroxybutyrate), for example,are also useful. Preferred poly(hydroxy acid)s are poly(glycolic acid),poly(DL-lactic acid) and poly(L-lactic acid), or copolymers ofpoly(glycolic acid and poly(lactic acid). In general, these materialsdegrade in vivo by both non-enzymatic and enzymatic hydrolysis, and bysurface or bulk erosion.

Biodegradable regions can be constructed from monomers, oligomers orpolymers using linkages susceptible to biodegradation, such as ester,peptide, anhydride, orthoester, and phosphoester bonds.

2. Non-Biodegradable Hydrophobic Polymers

Representative non-biodegradable, hydrophobic polymers that could beincorporated into the backbone of the comb copolymers includepolyalkylenes such as polyethylene and polypropylene, polychloroprene,polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate),polyvinyl halides such as poly(vinyl chloride), polysiloxanes,polystyrene, polyurethanes and copolymers thereof, polyacrylates, suchas poly(methyl (meth)acrylate), poly(ethyl (meth)acrylate),poly(butyl(meth)acrylate), poly(isobutyl (meth)acrylate),poly(hexyl(meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate) jointly referred to herein as “polyacrylates”), and copolymersand mixtures thereof The polymers include useful derivatives, includingpolymers having substitutions, additions of chemical groups, forexample, alkyl groups, alkylene groups, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art.

Preferred non-biodegradable polymers include ethylene vinyl acetate,polyacrylates, poly(chloroprene), and copolymers and mixtures thereof

C. Non-cell Binding Hydrophilic Side Chains

The non-cell binding side chains are preferably water-soluble when notattached to the backbone, and, more preferably, are non-ionic. Suitablepolymeric blocks include those prepared from poly(ethylene glycol),poly(ethylene oxide), partially or fully hydrolyzed poly(vinyl alcohol),poly(vinylpyrrolidone), and dextran. Preferably, the side chains aremade from poly(ethylene glycol), poly(ethylene oxide), or poly(acrylicacids).

The hydrophilic side chains may be intrinsically biodegradable or may bepoorly biodegradable or effectively non-biodegradable in the body. Inthe latter two cases, the side chains should be of sufficiently lowmolecular weight to allow excretion. The preferred molecular weightrange is below about 2000 Daltons, more preferably below 1000 Daltons,and most preferably, below about 500 Daltons. When the polymer ispolyethylene glycol, it is preferred that the number of ethylene oxidemonomeric units is between about 4 and 20.

When double-bond containing monomers are used to prepare the polymerbackbone, a preferred method for incorporating the hydrophilic sidechains is to use a hydrophilic macromonomer with a reactive double bondat one end which can be randomly incorporated during free radical orother addition polymerization. An example of such a macromonomer isPEG-methacrylate. The density of the non-cell binding, hydrophilic sidechains along the polymer backbone is controlled by controlling therelative amounts of the PEG-methacrylate or other suitablemacromonomeric unit used.

In those embodiments in which the side groups are end capped withcell-signaling ligands, appropriate functional groups, such as —NH₂,—OH, or COOH are included on the ends of the macromonomers.

D. Monomers with Reactive Functional Groups

In many of the embodiments described herein, the monomers used to formthe polymer backbone include only two reactive groups, both of which arereacted in order to form the polymer. For example, lactic acid includestwo reactive groups, a hydroxy group and a carboxy group. —OH is thepreferred reactive group. Although the ends of a polylactic acid polymerinclude a hydroxy group and a carboxy group, there are no reactivegroups along the backbone in the final polymer chain that can be used toform a comb copolymer.

Monomers which contain one or more additional reactive groups need to beincorporated into the polymer backbone, preferably in a random fashion,in order to form the comb-type copolymers when monomers that do notinclude these reactive groups are used to prepare the polymer backbone.Examples of these types of monomers are well known to those of skill inthe art.

The requirements for a suitable reactive monomer are that it can beincorporated in the growing polymer chain by participating in the sametypes of chemical reactions as the growing polymer chain. For example,when lactide is being polymerized using a Lewis acid catalyst, adepsipeptide (cyclic dimer of an amino acid) can be prepared fromlysine, in which the epsilon amine group is protected, for example, witha t-boc protecting group. The lysine is incorporated into the polymer,and the protecting group can be removed. The resulting amine groups arereactive with hydrophilic polymers which include leaving groups such astosylates, tresylates, mesylates, triflates and other leaving groupswell known to those of skill in the art.

Alternatively, the reactive monomer can include a leaving group that canbe displaced with a nucleophilic group on a hydrophilic polymer. Forexample, epichlorohydrin can be used during the polymerization step. Themonomer is incorporated into the polymer backbone, and the chloridegroup is present on the backbone for subsequent reaction withnucleophiles. An example of a suitable hydrophilic polymer containing anucleophilic group is a PEG with a terminal amine group. PEG-NH₂ canreact with the chloride groups on the polymer backbone to provide adesired density of PEG-ylation on the polymer backbone. Using thechemistry described herein, along with the general knowledge of those ofskill in the art, one can prepare polymer backbones which includesuitable leaving groups or nucleophiles for subsequent couplingreactions with suitably functionalized hydrophilic polymers.

E. Ligands for Controlling Cell Response

A number of molecules are known to promote cell adhesion. These can beamino acids, peptides or glycoproteins. Exemplary cell-binding ligandsinclude peptides possessing an Arginine-Glycine-Aspartic acid (RGD)amino acid sequence or a Tyrosine-Isoleucine-Serine-Arginine-Glycine(YISRG). The RGD sequence, present in proteins such as fibronectin, hasbeen shown to be active in promoting cell adhesion and growth (Massia,S. P. and Hubbell, J. A., J. Cell. Biol., 114:1089 (1991)).Incorporation of RGD sequences at the ends of the copolymer side chainsthus can enhance cell adhesion and growth. This is particularly usefulwhen a substrate is not adhesive, for example, a polyester to whichcells such as hepatocytes show poor adhesion, which is then modifiedwith the comb copolymer to promote cellular adhesion in a controlledmanner.

Biologically active molecules may also be incorporated into thecopolymer to promote the adhesion and growth of a particular cell typein vivo. Many growth factors are known and can be obtained fromcommercial sources such as Sigma Chemical Co, St. Louis, Mo., forexample, growth factors including epidermal growth factor, vascularendothelial growth factor, fibroblast growth factor, etc.

F. Relative Ratios of Comb Components

1. Ratio of Hydrophilic to Hydrophobic Units

The density of the hydrophilic side chains along the polymer backbonedepends in part on the molecular weight of the side chains. The totalpercent of the hydrophilic units to the hydrophobic units in the combcopolymers is between 20 and 60 percent by weight, preferably around 40percent by weight. For hydrophilic side chains with a molecular weightof about 350, the mole percent of backbone segments carrying hydrophilicside chains can be as high as about 30 percent. For hydrophilic sidechains with a molecular weight of about 2000, the mole percent can be aslow as about 2 percent.

The relevant consideration when determining an appropriate ratio ofhydrophilic to hydrophobic units in the comb copolymers is that theoverall polymer, when the hydrophilic side chains are not end-cappedwith cell-signaling ligands, has the defined non-cell binding propertiesand preferably is not water-soluble. A relatively high density of veryshort (MW 500 or less) hydrophilic side chains can provide the samedegree of resistance to cellular adhesion as a lower density of highermolecular weight (for example, a MW between 1500 and 2000) side chains.Those of skill in the art can adjust the molecular weight and density ofthe polymers taking these factors into consideration.

2. Density of Tethered Ligands

The non-cell binding side chains of the comb copolymers can beend-capped with cell-signaling chemical ligands in order to elicitspecific cell responses. Ligands such as adhesion peptides or growthfactors can be covalently or ionically attached to the ends of the sidechains using known chemistries to provide specific chemical signals tocells. A defined fraction of ligand-bearing side chains can be obtainedby using appropriate stoichiometric control during the coupling of theligands to the ends of the side chains, by protecting the end-groups onthose side chains which are not to be end-capped with ligands, or bycombinations of these approaches. For applications where it is desirableto cluster ligands on the length scale of nanometers or tens ofnanometers on a biomaterial surface, more than one ligand (on average)can be attached to each comb copolymer chain. In applications where itis desirable to incorporate two or more types of ligands in a singlecluster on a biomaterial surface on the size scale of nanometers to tensof nanometers, one or more of each of the ligand types (for example, anadhesion peptide and growth factor) can be attached to each combcopolymer chain (on average) using known chemistries. Presentation ofthe ligand (or ligands) at the surface can thus be tailored in terms ofoverall surface density by exploiting the multi-branch nature of thecomb molecule, in terms of local density, by the number of ligandsattached to the same comb. The ability of the polymers to controlcellular adhesion or other cell function can be adjusted by controllingthe density of the cell-signaling ligands presented at the surface.

II. Polymer Mixtures

A. Mixtures of Comb Copolymers

When adhesion peptides are coupled to the comb copolymer side chains,cells attach and spread readily on the comb copolymer surface. Theamount of cell spreading and proliferation on the surface therefore canbe controlled by mixing adhesion peptide-bearing comb copolymers withnon-cell binding comb copolymers, for example, so that less than 20%,more typically less than 2%, of the combs bear an adhesion peptide.Similarly, the spatial distribution of ligand clusters on thebiomaterial surface can be controlled by mixing non-cell binding combcopolymers with comb copolymers in which each chain on average has morethan one ligand attached to its side chains.

The size of the ligand clusters (i.e., the spatial area in which theligands are localized) is dictated by the characteristic size of theligand-bearing comb copolymer, and can be approximated from the combcopolymer's radius of gyration, R_(G), which can be calculated orexperimentally determined by one of ordinary skill in the art. The combcopolymer radius of gyration can range typically between nanometers andseveral tens of nanometers, depending on total molecular weight, lengthof side chains, and environment surrounding the polymer chain, forexample, other polymer chains or water molecules. Thus the size of theligand clusters, as well as the number and type of ligands per cluster,can be controlled by the synthesis conditions of the ligand-bearing combcopolymers. For example, a comb copolymer of R_(G) would have an areaper cluster of π R_(G) ². The number of clusters on the surface per unitsurface area (on average) can be controlled by the ratio ofligand-bearing to non-cell binding combs at the surface. To achieve asurface separation distance between ligand clusters of d, whered>2R_(G), the concentration of ligand-bearing combs should beapproximately φ=V_(chain)/(2R_(G)d²), where V_(chain) is the volumeoccupied by a single comb copolymer chain.

B. Mixtures of Comb Copolymers and Other Polymers

The copolymers described herein can be blended with other polymers thatdo not elicit controlled cell responses. In applications where it isdesirable to use the comb copolymer to modify the surface of a second,hydrophobic or non-cell regulating polymer, the comb copolymer can beadded in small quantities to the second polymer and processed to achievecomb segregation to the surface. Blends of the comb copolymers withother polymers include those containing between 1 and 99% by weight ofthe comb copolymers, preferably less than 20 wt % of the combcopolymers, and more preferably less than 10 wt % of the combcopolymers. Processing steps to achieve comb surface segregation includeheating the mixture under vacuum, in air, water, water vapor,supercritical CO₂ or other environment that favors the comb component atthe surface, at temperatures sufficiently above the glass transitions ofthe polymer components (the matrix polymer and the comb copolymeradditive) to provide mobility for achieving surface segregation. In thecase where the second polymer component is a semicrystalline polymer,the annealing temperature should be above the glass transition but belowthe melting point of the polymer, to ensure that the desired shape ofthe device is retained.

In preferred embodiments, surface segregation is achieved during astandard processing step in the manufacture of a biomedical device, suchas during an extraction, autoclaving or sterilization process. In otherembodiments, segregation is accomplished in an additional annealing stepin a controlled environment (water, etc), after device fabrication. Suchprocessing steps create a surface layer approximately 2R_(G) inthickness that contains almost exclusively the comb copolymer. Theobservable surface properties of such annealed mixtures aresubstantially identical to those of the pure comb copolymers. Inpreferred embodiments, the comb copolymer is miscible with the secondpolymer to avoid phase separation in the bulk device, which might leadto poor mechanical or optical properties.

In other cases, the localization of the comb polymer to the surface of adevice primarily comprised of a second, hydrophobic or non-cellregulating polymer can be accomplished during other steps of devicemanufacture. For example, precise placement of the comb copolymer at thesurface of a device made from a second polymer can be accomplished by3DP methods Likewise, differences in viscosity between the combcopolymer and a second polymer when blended together can be exploited tolocate the comb to the surface during melt extrusion of fibers, films orother devices. Porous or nonporous membranes, films, fibers or hollowfibers in which the comb copolymer resides at the surfaces can beprepared by phase inversion casting. In this method, a solution of thecomb copolymer, the second polymer, and a mutual solvent is cast into anaqueous-based coagulation bath to form the device. During the castingprocess, favorable interactions between the comb and the coagulationbath medium induce segregation of the comb copolymer to exteriorsurfaces of the film, fiber, or membrane. Cell-regulating microporousbiodegradable membranes useful as temporary barrier devices inwound-healing applications can be prepared in this fashion.Cell-regulating biodegradable sutures can similarly be prepared byspinning fibers from solution into an aqueous-based coagulation bath.Such surface-modified fibers can also be prepared from biodegradable ornonbiodegradable materials and fashioned into nonwoven fabric articlesfor biomedical applications including cell-regulating temporary barrierdevices and biofiltration devices. Hollow nanoporous fibers can beprepared which have cell-regulating interior surfaces. By encapsulatingcells in a portion of such a fiber, a long-term drug delivery implantcould be prepared which secretes desirable products of cells inquantities regulated wholly or in part by tethered signals on the fiberinner surface. Cell-regulating biodegradable microporous scaffolds witha surface excess of comb copolymers can be prepared by freeze-dryingmethods by choosing a sublimating solvent which has preferentialaffinity to the comb copolymer component as compared to the secondpolymer component which forms the bulk of the device.

In all cases described above where comb copolymers are used inconjunction with a second polymer to prepare a device, the combcopolymers can be non-cell binding combs, ligand-bearing combs, or amixture of these to achieve a desired cell response as previouslydescribed. The observable surface properties of the device aresubstantially identical to those of the comb copolymer or comb copolymermixture itself.

III. Latexes Prepared with Comb Copolymers

A. Latex Synthesis

A further method by which the comb copolymers can be used forcontrolling cell response in biomedical applications is through thepreparation of polymer latexes that incorporate the comb copolymers onthe latex particle surfaces. Such latexes can be prepared by dispersionor emulsion polymerization methods in a water-containing medium, usingthe comb copolymers as a stabilizing agent. The polymerization isachieved by dissolving the desired monomer, comb stabilizer andinitiator in a water-containing medium. The polymer is initiated, forexample, by applying heat to the solvent. The dispersion medium is agood solvent for the comb copolymer but a poor solvent for the growingpolymer. The hydrophobic comb backbone is chosen to be compatible withthe polymer being synthesized, and thus anchors to the surface of thegrowing polymer particles, while the hydrophilic side chains stabilizethe particles against flocculation. Upon completion of the latexsynthesis, the resulting latex particles are in the range of 0.1 to 10μm in size, typically dispersed at 20-70% polymer solids by weight inthe dispersion medium. These systems can be employed in a variety ofways to control cell response through the comb copolymers that remainanchored to the particle surfaces.

Polymers which might be synthesized as latex particles fornon-biodegradable applications include polyvinyl ethers, polyvinylesters such as poly(vinyl acetate), polyvinyl halides such as poly(vinylchloride), polystyrene, and polyacrylates, such as poly(methyl(meth)acrylate), poly(ethyl (meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), and poly(octadecyl acrylate), and copolymersand mixtures thereof, as well as useful derivatives of these polymers,including polymers having substitutions, additions of chemical groups,for example, alkyl groups, alkylene groups, hydroxylations, oxidations,and other modifications routinely made by those skilled in the art.

Polymers which might be synthesized as latex particles for biodegradableapplications include poly(amino acids), poly(anhydrides),poly(orthoesters), and poly(phosphoesters), polylactones such aspoly(epsilon-caprolactone), poly(delta-valerolactone),poly(gamma-butyrolactone)and poly (beta-hydroxybutyrate), andpoly(hydroxy acid)s such as poly(glycolic acid), poly(DL-lactic acid)and poly(L-lactic acid), or copolymers of poly(glycolic acid andpoly(lactic acid).

B. Latex Films

Films or coatings can be prepared from the latex dispersions by usualmethods such as dipping, brushing, rolling or casting the latex onto anysurface. For coatings applied to permanent implants to control cellresponse, non-biodegradable latex particles prepared withnon-biodegradable comb stabilizers are preferred. For applications wherea biodegradable film is preferred, biodegradable latexes can be preparedusing comb stabilizers with biodegradable backbones. Opaque coatings maybe prepared that elicit controlled cell response by employing any of thestandard coating methods used to form latex films, such as those justmentioned. Alternatively, by heat-treating films at a temperature wellabove the glass transition of the polymer particles, the particles willcoalesce into a smooth, transparent film in which the comb copolymersreside at the surface. The comb copolymers remain localized at thesurface upon coalescence due either to an energetic tendency to remainat the surface, or because there is insufficient mobility for combdiffusion into the bulk of the coalesced latex film, for example, if thefilm is cooled below its glass transition shortly after coalescence.

The latex films exhibit surface properties of the comb copolymersthemselves, but have the advantages that only small quantities of thecomb copolymer are required (typically below 1 wt % of the total latex),coatings can be easily applied from water-based suspensions, and thefilm-forming properties can be tailored to adhere to the substrate byjudicious choice of the film-forming polymer. For example, an acryliclatex stabilized by non-cell binding comb copolymers could be used toprepare transparent acrylic coatings on acrylic intraocular lenses inorder to render them resistant to cell attachment, and hence lesssubject to clouding over time. Acrylic latexes could also be used inapplications where controlled cell response is desired at the surface ofpermanent metal, glass or ceramic implants or other devices, includingcell culture apparatus, since a high degree of adhesion is often foundbetween oxide surfaces and acrylic polymers. For polystyrene cellculture plates or other apparatus, a cell-regulating PS latex could beused to prepare a transparent, cell-regulating coating in the mannerdescribed above.

In all cases described above where latexes are stabilized by combcopolymers, the comb copolymers might be non-cell binding combs,ligand-bearing combs, or a mixture of these to achieve a desired cellresponse as previously described above. Alternatively, mixed latexdispersions can be used to prepare films that contain clustered ligandregions on a surface of sizes from 0.1 to 10 micrometers. This can beachieved by mixing together dispersions of latex particles coated withnon-cell binding combs and those coated with ligand-bearing combs andcreating films of these mixed dispersions as described above. The sizeof the ligand clusters is approximately the diameter of the latexparticles coated with ligand-bearing combs, while the number of clusterson the surface per unit surface area can be controlled by the ratio ofligand-bearing to non-cell binding latex particles in the mixeddispersion.

IV. Polymer Preparation

Methods for preparing hydrophobic polymers including reactive monomericunits are known. Typical reactions are ring opening polymerization (formonomers such as lactide, glycolide, and other cyclic monomeric units),free radical polymerization (for double bond-containing monomeric unitssuch as methyl methacrylate), and anionic or other additionpolymerizations.

The monomers used to prepare the hydrophobic polymer backbone, forexample, lactide, glycolide, caprolactone, and trimethylene carbonate,can be reacted with various polymerization initiators, for example,alcohols such as ethylene glycol and ethanol, water, and amines, in thepresence of a suitable catalyst such as a Lewis acid, as described, forexample, in Kricheldorf, H. R. in Models of Biopolymers by Ring-OpeningPolymerization, Penczek, S., Ed., CRC Press, Boca Raton, 1990, Chapter1; Kricheldorf, H. R. α-Aminoacid-N-Carboxy-Anhydrides and RelatedHeterocycles, Springer-Verlag, Berlin, 1987; and Imanishi, Y. inRing-Opening Polymerization, Ivin, K. J. and Saegusa, T., Eds.,Elsevier, London, 1984, Volume 2, Chapter 8.

The cell-binding polymer side chains grafted onto the polymer backboneare preferably hydrophilic polymers, such as polyethylene glycol,polyethylene oxide, polyacrylic acid, dextran and mixtures thereof,which can be modified to include reactive functional groups such asamino, carboxylic acid, halo, sulfide, guanidino, imidazole and hydroxylgroups. These groups can react with various reactive groups on thepolymer backbone in routine nucleophilic displacement reactions to graftthe hydrophilic polymers to the backbone. The side chain polymers can beend-capped with cell binding ligands through standard covalent or ioniccoupling reactions.

V. Surface Coatings and Devices incorporating Comb Copolymers

Numerous methods can be used to apply the comb copolymers, combcopolymer mixtures, or mixtures of comb copolymers and other polymers tosurfaces. These methods include dip coating, spray coating, brushcoating, roll coating, or spin casting a film onto the substratefollowed by mild heating to promote adhesion to the surface. Solid freeform processes such as 3DP, or freeze drying methods could be used tocreate complex three-dimensional structures, including porousstructures. In all of these processing approaches a suitablecrosslinking agent might be incorporated to enhance the mechanicalrigidity of the coating or device.

In applications where mixtures of comb copolymers with other polymersare desirable, processing steps to achieve comb surface segregationinclude heating the mixture under vacuum, in air, water, water vapor,supercritical CO₂ or other environment that favors the comb component atthe surface, at temperatures sufficiently above the glass transitions ofthe polymers to provide the combs with the necessary mobility. In thecase where the second polymer component is a semicrystalline polymer,the annealing temperature should be above the glass transition but belowthe melting point of the polymer, to ensure that the desired shape ofthe device is retained.

Surface segregation could be achieved preferably during a standardprocessing step in the manufacture of a biomedical device, such asduring an extraction, autoclaving or sterilization process, or could beaccomplished in a separate annealing step after the device has beenmanufactured. This type of processing creates a surface layer on thedevice that contains almost exclusively the comb copolymer. In othercases, the localization of the comb polymer to the surface of a deviceprimarily comprised of a second polymer can be accomplished during othersteps of device manufacture. For example, differences in viscositybetween the comb copolymer and a second polymer when blended togethercan be exploited to locate the comb to the surface during melt extrusionof fibers, films or other devices. Porous or nonporous membranes, films,fibers or hollow fibers in which the comb copolymer resides at thesurfaces can be prepared by phase inversion casting. In this method, asolution of the comb copolymer, the second polymer, and a mutual solventis cast into an aqueous-based coagulation bath to form the device.During the casting process, favorable interactions between the comb andthe coagulation bath medium induce segregation of the comb copolymer toexterior surfaces of the film, fiber, or membrane. Cell-regulatingmicroporous biodegradable membranes useful as temporary barrier devicesin wound-healing applications can be prepared in this fashion.Cell-regulating biodegradable sutures can similarly be prepared byspinning fibers from solution into an aqueous-based coagulation bath.Such surface-modified fibers can also be prepared from biodegradable ornonbiodegradable materials and fashioned into nonwoven fabric articlesfor biomedical applications including cell-regulating temporary barrierdevices and biofiltration devices. Hollow nanoporous fibers can beprepared which have cell-regulating interior surfaces. By encapsulatingcells in a portion of such a fiber, a long-term drug delivery implantcould be prepared which secretes desirable products of cells inquantities regulated wholly or in part by tethered signals on the fiberinner surface. Cell-regulating biodegradable microporous scaffolds witha surface excess of comb copolymers can be prepared by freeze-dryingmethods by choosing a sublimating solvent which has preferentialaffinity to the comb copolymer component as compared to the secondpolymer component which forms the bulk of the device.

V. Biomedical Applications

The comb-type copolymers described herein may be used in a variety ofbiomedical applications, such as in scaffolds and supports for cellgrowth in tissue engineering, coatings for biomedical implants such asintraocular lenses or other permanent implants made from polymeric,metal, glass, or ceramic materials, and coatings for cell cultureapparatus such as cell culture plates, pipets, etc.. The comb-typecopolymers may be used for modifying the surface properties of sutures,temporary barrier films or fabrics in wound-healing applications,artificial hearts and blood vessels, catheters, filters for blood orother body fluids, and targeted controlled-release drug deliveryvehicles and encapsulated cell drug delivery systems. The materials arepreferably biodegradable when used for tissue engineering, woundhealing, and targeted drug delivery applications, and are preferablynon-degradable when used to modify implants, cell culture apparatus,filtration devices, and other devices intended for long term use orimplantation.

A. Tissue Engineering

For use in tissue engineering applications, the comb copolymers may bederivatized by the attachment to the ends of the hydrophilic side chainsbiologically active molecules that promote favorable cell-polymerinteractions, such as cell adhesion molecules and growth factors.Matrices suitable for seeding or ingrowth of cells can be formed whichincorporate the comb copolymers, or a matrix formed of a material suchas stainless steel, collagen, or another polymer can be coated with thecomb copolymers. The matrix is then either seeded with cells andimplanted, or the matrix implanted for tissue ingrowth to occur. Thesematerials can be tailored to fit the particular needs of a variety ofcell types through changes in the type and density of cell adhesionpeptides attached to the copolymers. Cell types which can be seeded onthe matrices include parenchymal cells such as hepatocytes,uroendothelial cells, skin cells, muscle cells, nerve cells and boneand/or cartilage forming cells. Normal cells, fetal cells or geneticallyengineered cells can be seeded onto the matrices.

B. Drug Delivery and Imaging

The comb copolymers also may be formed into matrices for use as drugdelivery systems or for imaging purposes. Biodegradable latexes coatedwith the comb copolymers be can be used for targeted delivery of atherapeutic, prophylactic or diagnostic agent. Hollow nanoporous fiberscan be prepared which have cell-regulating interior surfaces comprisedof comb copolymers or comb copolymer mixtures. By encapsulating cells ina portion of such a fiber, a long-term drug delivery implant could beprepared which secretes desirable products of cells in quantitiesregulated wholly or in part by tethered signals on the fiber innersurface.

For use in drug delivery, a therapeutic or prophylactic agent, such asan amino acid, bioactive peptide or protein, carbohydrate, sugar, orpolysaccharide, nucleic acid or polynucleic acid, synthetic organiccompound, or metal may be attached to through the end groups of thehydrophilic side chains of the comb copolymer using methods available inthe art. The comb copolymers may be modified to increase the level ofthe incorporated agent. Agents which provide greater stability for theagent to be delivered may be covalently or ionically attached to thecopolymer. The comb copolymers may be functionalized with a specificbinding moiety, e.g, an antibody, which targets the latex particle fordelivery to a particular site within the body Hydrophilic, hydrophobic,acidic, basic or ionic side chains also may be attached to thecopolymers to expand their use as delivery devices for drugs. Matricesof the modified drug-containing comb copolymer may be administered to ananimal orally or parenterally to deliver the drug to the animal in vivoat a site in the animal where it is needed.

Diagnostic agents include radioactive materials, fluorescent materials,enzymatic materials, gases, and magnetic materials.

C. Use of the Materials to Provide Cell Repulsive Surfaces

It is often desirable to minimize cell and tissue interactions withbiomedical implants, such as intraocular lenses. These interactions areminimized when the surface of an implant is coated with the non-cellbinding copolymers. It is preferred that the copolymer be non-degradablein some applications. For example, when intraocular lenses areimplanted, they are intended to remain in place for extended periods oftime and biodegradability is to be avoided.

A preferred non-biodegradable polymeric material is a copolymer of analkyl acrylate (i.e., methyl methacrylate) and PEG-methacrylate. Apreferred method to place this coating at the surface is through theformation of a latex film.

The present invention will be further understood by references to thefollowing non-limiting examples, in which the following materials andequipment were utilized.

EXAMPLE 1 Preparation, Processing, and Evaluation of Biodegradable CombCopolymers and Their Blends

Comb Polymer Synthesis

Lactide, epichlorohydrin, poly(ethylene glycol) methyl ether (MPEG,M_(W)˜350 g/mole), poly(ethylene glycol) (PEG, M_(W)˜400 g/mole), andanhydrous toluene (all from Aldrich Chemical Co.) were used as received.

Tetrahydrofuran (Aldrich Chemical Co.) was distilled prior to use.Lactide and epichlorohydrin (Aldrich Chemical Co ) were copolymerized byring opening polymerization (Shen et al. J. Polym. Sci., Polym. Chem.Ed., 31:1393 (1993)) at 100° C. in toluene with a trioctylaluminum-water catalyst. The in situ AlOct₃:0.5 H₂O catalyst wasprepared using a modification of a literature procedure. Briefly, AlOct₃(25 wt % in hexane, Aldrich Chemical Co.) and distilled THF were stirredin a sealed flask under nitrogen and allowed to equilibrate at −68° C.in a dry ice/acetone bath. H₂O was added to the mixture to give a 1:0.5molar ratio between AlOct₃ and water. The mixture was stirred vigorouslyat −68° C. for 15 minutes, then removed from the dry ice bath andallowed to return to room temperature over 30 minutes. The catalystsolution was then injected into a sealed reaction flask containinglactide, epichlorohydrin, and toluene under nitrogen and allowed toreact 16 hours at 100° C. The resulting LA-EO copolymer was purified byrepeated precipitation in petroleum ether.

Grafting of MPEG and PEG to the LA-EO copolymer was performed by phasetransfer catalysis (Ober, Makromol. Chem., Macromol. Symp.35:36-87(1990)) reacting the terminal hydroxyls of the ethylene glycolchains with the pendant chlorine groups of the backbone copolymer. TheLA-EO copolymer was dissolved in methylene chloride. PEG, MPEG, and pH 8aqueous NaHCO₃ were then added with vigorous stirring. The mixture wasallowed to react overnight. Unreacted glycols were removed from thepolymer by repeated precipitations in methanol. The final non-cellbinding comb copolymer had a molecular weight of approximately 40,000Daltons, was insoluble in water, and incorporated approximately 40% byweight hydrophilic PEG side chains.

A pentamer amino acid sequence, Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP fromGibco, referred to herein as RGD), was used to create adhesionligand-bearing comb copolymers by tethering the RGD to functionalizedends of PEG side chains. RGD interacts specifically with receptors knownas integrins on the surface of cell membranes, and RGD-integrin couplingmediates adhesion of cells to their surroundings in vivo. RGD wascoupled to the non-cell binding comb copolymers via primary amines usingknown tresyl chloride chemistry (Obel, et al. J. Polym. Sci., Polym.Lett. Ed. 23:103 (1985)). Comb copolymers were activated with tresylchloride groups in solution and stored at −20° C. until use. RGD wascoupled to the combs by immersion in PBS solutions of RGD (25 μg/mL, pH7.4) at 5° C. for 3 hours. Systems were multiply rinsed with PBS toremove unreacted RGD.

Film Processing

Mixtures of the activated comb copolymers and the non-cell binding combcopolymers were prepared in various ratios, and cast from solution intoluene onto glass slides. Films were subsequently dried under vacuumfor 24 hours to remove residual solvent. RGD was subsequentlysubsequently coupled to the exposed activated comb polymers at thesurface of the films in the manner described above. For mixturescontaining 100 wt %, 25 wt % and 5 wt % tresyl-activated comb copolymer,surface RGD densities of 9.5 pg/cm², 2.5 pg/cm², and 0.5 pg/cm² wereachieved, respectively.

Mixtures of polylactide, PLA, homopolymer and small amounts of thenon-cell binding or RGD-bearing comb copolymers (10 wt % or lessrelative to PLA) were dissolved in toluene, and cast as films on glass.Films were subsequently dried under vacuum for 24 hours to removeresidual solvent. Some of the comb/PLA films were subsequently annealed96 hours in a 70° C. water bath. X-ray photoelectron spectroscopystudies showed significant enrichments of the comb copolymer at thesurface of annealed blends (˜60% by volume comb copolymer at the surfacefor a 10% bulk concentration). Advancing/receding contact anglemeasurements similarly indicate that the annealed blend films havesubstantially lower water contact angles than PLA and exhibit a largehysteresis indicative of PEG side chain reorientation/hydration at thesurface when in contact with water.

Cell Culture

NR6 fibroblasts were cultured in serum-containing media onto the mixedcomb films, the comb/PLA blends, and a PLA control film. Polymer filmswere first sterilized by immersion in ethanol Wild-type NR6 fibroblastswere seeded on polymer film surfaces in Modified Eagle's Mediumsupplemented with fetal bovine serum. Cells were cultured for 24 hours,media was aspirated, and fresh media applied before phase contrastphotomicrographs were taken.

Films of the non-cell binding combs were completely resistant to celladhesion over 24 hour time periods, even in the presence ofserum-supplemented medium. This is believed to be due to the formationof a dense hydrated layer of PEG side chains at the film surface. In themixed comb films, increasing surface densities of RGD increased theadhesion and spreading of cells on the film surface. Variation in theweight fraction of RGD-coupled combs in films with unfunctionalizedcombs from 0 to 100% allowed a change in the adhesive response of cellsto the surfaces. At 0% RGD-combs, no cells adhered, at 5% RGD-combscells stuck but retained a rounded morphology, and at 100% RGD-combscells were strongly adhered and spread on the surfaces.

This result demonstrates that comb mixtures can provide some level oftunable ligand presentation and control over cell adhesion. TheRGD-bearing surfaces supported cell adhesion and spreading even in theabsence of serum. In addition, soluble RGD added to media abrogated thespreading of cells and detached them from the surfaces. These resultsindicate that the effects seen are due to specific interactions betweencellular integrins and RGD and not interactions between integrins andserum proteins adsorbed on RGD.

On the surface of annealed PLA/non-cell binding comb blends, no celladhesion was found due to the formation of the comb-enriched surfacelayer which resists protein adsorption. By comparison, cell adhesion wasobserved on unmodified PLA and, to a lesser degree, on the unannealedblend, which both allowed cells to stick and spread in an uncontrolledfashion. Cell culture studies on annealed PLA/RGD-bearing comb blendsshowed significant controlled cell attachment through the RGD ligands,even in the absence of serum.

Modulation of the degree of cell adhesion was also demonstrated in cellculture experiments with primary rat hepatocytes. The cells were platedat a density of 30,000 cell/cm² on substrates containing either 1% or100% RGD-bearing comb in a 10% comb/PLA blend film, prepared andannealed as described above. Hepatocytes remain highly spread onsubstrates containing 100% RGD combs, but aggregate and assume aspheroidal morphology on blends in which only 1% of the combs wereRGD-bearing.

Example 2 Preparation of Biodegradable Devices from Comb/PLA Blends

Porous Scaffold

Biodegradable PLA/comb copolymer microporous scaffolds that might beused as substrates for tissue engineering applications were prepared byfreeze drying solutions of 10% wt/vol polymer in dioxane.Blends-containing 10 wt % of the biodegradable, non-cell binding combcopolymer and 90 wt % PLA were dissolved in dioxane and frozen in liquidnitrogen, causing the phase separation of the polymer and solvent. Uponsublimating the dioxane, a porous biodegradable foam was obtained, whichcould be further treated, for example, by autoclaving or heat-treatingin deionized (Dl) water at 90° C., to achieve a high coverage of thecomb copolymer on the pore exterior surfaces.

Temporary Barrier Membrane

Biodegradable PLA/comb copolymer microporous membranes that might beused as temporary barriers in wound healing applications were preparedby phase inversion casting from solutions of 10-20% polymer inN,N-dimethylformamide (DW). Blends containing 10 wt % of thebiodegradable, non-cell binding comb copolymer and 90 wt % PLA weredissolved in DMF and cast using a doctor blade onto a cleaned glasssubstrate. The substrate was immediately immersed in a bath of deionized(DI) water at 90° C. to create a porous membrane structure during theprecipitation of the insoluble polymer. Once formed, the membranes wereremoved and rinsed in a second DI bath at 90° C. to remove trace solventimpurities.

Example 3 Preparation and Evaluation of Non-Biodegradable CombCopolymers and Their Mixtures

Comb Synthesis

Non-biodegradable comb polymers were synthesized by free radicalpolymerization of methyl methacrylate (MMA) with either methoxypoly(ethylene glycol) methacrylate (MPEGMA) or poly(ethylene glycol)methacrylate (PEGMA) or a mixture of these initiated in toluene at 70°C. by azo(bis)isobutyronitrile. After 12-16 hours the reaction wasterminated, and the polymer precipitated in petroleum ether. Theresulting comb polymer has a PMMA backbone with PEO side chains nearlyrandomly distributed along the backbone, and a molecular weight ofapproximately 20,000 g/mole. The PEGMA macromonomers provide side chainsend-capped with a hydroxyl group which can be derivatized for covalentlinkage of the peptides, while the MPEGMA units provide non-reactivemethoxy-terminated PEG side chains. Combs containing ˜40% PEG sidechains by weight are insoluble in water but form very hydrophilic,protein- and cell-resistant surfaces, and thus are considered non-cellbinding.

To obtain adhesion ligand-bearing non-biodegradable combs, the RGDpeptide was attached to hydroxyl end groups of the PEG side chains. Thecombs were dissolved in anhydrous tetrahydrofuran (THF), followed byaddition of triethylamine and tresyl chloride, and reacted for 90minutes. The activated polymer was recovered by precipitation inanhydrous methanol, and stored at −70° C. until use. RGD was coupledthrough primary amines to the activated combs by first dissolving thecombs in dry THF, followed by addition of peptide solution (1 mg/mLGRGDSP in phosphate buffered saline (PBS)) at a ratio of 10:1 THF:PBS.Coupling was allowed to proceed with stirring for 3 hours at 5° C. Theresulting RGD-comb polymer was recovered by precipitation/washing withdeionized water.

Film Preparation and Cell Culture

Films for cell culture were prepared by spin-coating the comb polymersonto glass substrates from anhydrous toluene. Purely cell-resistantsurfaces were prepared by spin-coating solutions of the non-cell bindingcombs, while ligand-bearing surfaces were made by spin-coating solutionscontaining both non-cell binding combs and RGD-bearing combs.

NR6 fibroblasts transfected with the wild-type human epidermal growthfactor receptor (WT NR6) were cultured in modified Eagle's medium alpha(MEM-α) supplemented with 7.5% fetal bovine serum, L-glutamine,non-essential amino acids, sodium pyruvate, penicillin-streptomycin, andgentamycin antibiotic. Cell were seeded at 20,000 cells/cm² onto combcopolymer films for 24 hours, followed by aspiration to removeunattached cells and application of fresh medium. Morphology/adhesion ofcells to films was then assessed using a Zeiss Axiovert 100 phasecontrast microscope. No cell adhesion was observed on films of non-cellbinding combs. In contrast, films of the RGD-bearing combs supportedadhesion and produced cell morphologies comparable to that observed onfibronectin.

Example 4 Preparation and Evaluation of EGF-Tetliered Comb Films

Film Preparation

To obtain non-biodegradable combs with tethered epidermal growth factorligands, non-cell binding combs with PMMA backbones and PEG side chainswere prepared as described in Example 3. EGF was attached to hydroxylend groups of the PEG side chains by first activating the side chainswith tresyl chloride following the procedure described above.

Films of the tresyl-activated comb were spin-coated at 1000 rpm from0.01 g/ml toluene solutions. Films were subsequently dried under vacuumto remove residual solvent, then sterilized by UV exposure for 1 hour.EGF was coupled to surfaces by incubating 5 μg/ml sterile PBS solutions(100 mM phosphate) of EGF on the films for 3 hours at 5° C. Solutionswere aspirated and samples were blocked with 100 mM pH7 sterile trissolutions 1 hour at 20° C. Controls were hydrolyzed in the presence oftris, thereby capping all the tresyl sites with tris instead of EGF. Onehydrolyzed control was exposed to an EGF solution under conditionssimulating the EGF coupling step to check for nonspecific adosrption ofEGF: Samples were multiply rinsed with sterile PBS. This protocolprovided 1.0±10.3 ng/cm² tethered EGF on the film surface.

Cell Culture

PC12 cells were seeded (medium: RMPI 1640 with 5% FBS, 10% horse serumheat-inactivated donor herd, and supplemented withpennicilin-streptomycin) on surfaces and cultured 3 days. To keep thePC12 cells attached to the surface in this experiment, prior toculturing, surfaces were exposed to 0.5 mg/ml rat tail collagensolutions overnight at 5° C. PC12s are an adrenyl tumor cell type whichdifferentiates into a neuronal phenotype under certain conditions. Thisdifferentiation is similar to that of neuronal cells in general,morphologically characterized by the formation and extension ofneurites. PC12 cells cultured in the presence of soluble EGF arereported to undergo a morphological change induced by the growth factorsignal- cells round up on adhesive surfaces, likely due either todown-regulation of integrins or changes in integrin-ECM affinity inducedby EGF signals. The EGF-bearing comb films are non-adhesive to cells andthe collagen treatment leads to only weakly cell-adhesive surfaces. PC12cells were cultured as described for several weeks. At 3 days initialevidence of differentiation was observed, which became very clear aftertwo weeks. No differentiation was observed on controls

Example 5 Preparation of Surfaces Presenting Multiple Ligand Types

Non-cell binding comb copolymers with PMMA backbones and methoxy- orhydroxyl-terminated PEO side chains were prepared as described inExample 3. The combs were subsequently used to create substrates whichpresent co-tethered epidermal growth factor (EGF) and RGD. First,RGD-bearing combs were prepared in the manner described in Example 3.The RGD-bearing combs were solvated in THF along with non-cell bindingcombs activated with tresyl chloride, and films were cast onto cleanedglass substrates using standard spin-coating procedures. Films weredried under vacuum for 24 hrs to remove remaining solvent. The substratewas then exposed to an EGF solution, enabling the covalent attachment ofEGF to the activated comb side chains at the surface through theterminal amine group of the EGF. Solutions of 10 ng/mL EGF in PBS wereincubated on surfaces containing the activated combs mixed with the RGDcombs, or with controls containing unactivated combs. The amount of EGFcovalently linked to the substrates under these conditions was 8.5±1.5ng/cm². For comparison, maximum DNA synthesis response in primary rathepatocytes cultured on tethered EGF occurred at a density of less than1 ng/cm² (1000 EGF molecules/μm²) and the approximate density ofreceptors on the cell surface of hepatocytes or WT NR6 is 100-400molecules/μm². Thus the amount of EGF which can be covalently linked onthe RGD-bearing substrate is sufficient for influencing cell response.Further, the amount of non-specifically adsorbed EGF on the combsurfaces, 0.9±0.3 ng/cm², is negligible relative to the amount that iscovalently coupled. WT NR6 fiberblasts were cultered for 24 hours aspreviously described on the EGF/RGD substrates. Cells were observed toadhere and spread on the mixed ligand surface.

Example 6 Comb Copolymer-Stabilized Latexes

Comb Synthesis

Comb polymer stabilizer was synthesized free-radically in solutionMethyl methacrylate (MMA), methoxy poly(ethylene glycol) methacrylate(MPEGMA), and poly(ethylene glycol) methacrylate (PEGMA) were added tobenzene in equal weight fractions of the two PEG macro-monomers, for atotal monomer concentration of 0.6M. Azo(bis)iso-butyronitrile was addedat a molar ratio of 20:1 [monomer]:[initiator]. The solution wasdegassed under nitrogen 15 minutes, followed by polymerization at 60° C.for 16 hours. The comb polymer was purified by repeated precipitation inpetroleum ether. In order to obtain latex beads with protein-resistantsurfaces, the ratio of PEGMA/MPEGMA units to MMA units in the combstabilizer copolymers was first optimized. Initial studies found thatcombs containing 40 wt % of the PEGMA/PEGMA units formed films that werecell resistant in the presence of serum and simultaneously resistant todissolution in water-based media. Combs of this composition were solublein 50/50 water/ethanol, and thus served as an ideal stabilizer forpreparation of the polymer latexes. Combs with greater PEG fractions(˜50 wt % or more) were water soluble over time. The comb stabilizer hada total molecular weight, prior to peptide attachment, of approximately23,000 Daltons.

To obtain adhesion ligand-bearing latexes, RGD-bearing combs were firstprepared by solution coupling GRGDSP (Gibco) to the ends of the PEGMAunits of the comb. Coupling was accomplished through the reaction oftresyl chloride-activated combs and the N-terminal amine of the peptide.The hydroxyl ends of the PEGMA units of the comb were activated byreaction with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride)in tetrahydrofuran. The comb copolymer (150 mg) was dissolved in 25 mldry THF at 5° C. Triethylamine (200 μl) and tresyl chloride (250 μl )were added and the reaction was allowed to proceed 3 hours. Theactivated polymer was then recovered by filtration and precipitation inpetroleum ether. GRGDSP peptide was coupled to the activated polymer byadding 150 μl GRGSP solution (1 mg/ml in pH 7.4 phosphate bufferedsaline) to 2.5 ml of activated comb solution (0.02 g/ml in THF) at 5° C.and stirring for 3 hours. The RGD-coupled comb was recovered byovernight precipitation in deionized water. Amounts of peptide coupledwere determined by a colorimetric assay (microBCA, Pierce Chemical Co.).RGD content was found to be 0.3 wt % (1 RGD peptide per ˜10 comb polymermolecules).

Acrylic Latex Syntheses

Methacrylate- and acrylate-based polymer latexes were synthesized bydispersion polymerization employing the comb polymers as stabilizingagents. Latexes of four different compositions were prepared in thisstudy: pure poly(methyl methacrylate), poly(methyl methacrylate-co-butylacrylate), poly(ethyl methacrylate-co-methyl acrylate), and poly(ethylmethacrylate-co-butyl methacrylate). In addition, one cell-interactivepoly(methyl methacrylate) latex was prepared using the RGD-combstabilizer. Comb stabilizer was dissolved in a 1:1 mixture by volume ofethanol and water, followed by addition of methacrylate/acrylatemonomers and 0.57 g ammonium persulfate. Reactions were allowed toproceed 18 hours at 60° C. with stirring. Reactions began as one phase,clear solutions, and became opaque white dispersions duringpolymerization. After completion of the syntheses, all latexes werepurified by repeated centrifugation and redispersion in water/ethanol.Suspensions were stable over greater than 24 hour periods and could beresuspended after extended storage via ultrasonic mixing. All latexeswere ultrasonically treated for at least 30 minutes prior to use.Molecular weights of the polymers comprising the latex particles rangedfrom approximately 400,000 Daltons to 1 millon Daltons. Glass transitiontemperatures of the particles ranged from −26° C. to 105° C., dependingon the monomer constistuents used in the polymerization.

Morphology of the latex beads was assessed by examining beads cast onsubstrates using a JEOL 6320 field emission scanning electron microscopeoperating at a 4.0 kV accelerating voltage. Samples were shadowed withgold prior to imaging. Average particle diameters were measured from SEMmicrographs, with at least 300 particles measured for each sample.Average particle sizes ranged from 0.2 to 1.8 micrometers. All of thelatexes had size polydispersities below 1.06. In each case, the combstabilizer comprised below 1 wt % of the total latex bead composition.

Latex Film Preparation and Characterization

Films were prepared from the latex suspensions by spin-coating theparticles (0.02-0.03 g/ml in water/ethanol) at 1000 rpm onto cleanedglass substrates. To form contiguous films from the cast particles,short heat treatments (30-60 seconds) were applied to the samples by aheat gun set at 800-900° C. Coalescence of the particles was confirmedby examining the surfaces in a light microscope. For cell culture andcontact angle experiments, poly(methyl methacrylate) homopolymer (not alatex) served as a control substrate. PMMA (Polysciences, 68 K g/mole,M_(W)/M_(N)=1.07) films were spincoated from a 0.03 g/ml toluenesolution onto clean glass coverslips at 1000 rpm, followed by drying invacuo at 70° C. 24 hours.

Contact angles of water on coalesced latex film surfaces, films of thenon-cell binding comb, and on the PMMA control film were measured usinga VCA2000 video contact angle system (AST Inc.). Advancing/recedingcontact angles were measured by capturing digital images of deionizedwater droplets placed by syringe on virgin surfaces and measuring anglesfrom the images. In all cases but the control, the advancing contactangles are seen to be relatively constant and independent of dropvolume, while the receding angles show significant changes in contactangle with drop size. All of the latex films showed hysteresis of 25° ormore in these measurements, while pure PMMA displayed only a ˜10°change. Though contact angle hysteresis can occur for a number ofreasons, the likeliest explanation for the contact angle hysteresisobserved here is the reorganization/hydration of the PEG side chains atthe surface of the films upon wetting. That the comb copolymer is notwater-soluble was confirmed by ellipsometry measurements of dried latexand comb film thicknesses before and after water immersion, which showedno detectable loss of polymer. These results provide a strong indicationthat the comb stabilizer remains at the surface once the latex particlescoalesce into a homogeneous film.

Cell Culture

All cell culture reagents were purchased from Gibco. NR6 fibroblaststransfected with the wild-type human epidermal growth factor receptor(WT NR6) were cultured in modified Eagle's medium alpha (MEM-α)supplemented with 7.5% fetal bovine serum, L-glutamine, non-essentialamino acids, sodium pyruvate, penicillin-streptomycin, and gentamycinntibiotic.

Cell attachment studies were performed by seeding 20,000 cells/cm² ontothe non-cell binding comb copolymer films, coalesced PMMA latex films,and two controls: tissue culture polystyrene (TCPS) and pure PMMA films.Cell were seeded in 1.5 ml serum-containing growth medium for 24 hours,followed by aspiration to remove unattached cells and application offresh medium. Morphology/adhesion of cells to latex films was thenassessed using a Zeiss Axiovert 100 phase contrast microscope.

After 24 hours, cells are attached and spread on both controls,presumably via protein layers adsorbed onto these surfaces. However, thePEG side chains of the comb copolymer stabilizer provide complete cellresistance for the comb film under these stringent conditions. Likewise,the PMMA latex film presents a surface with essentially equivalentcell-resistant capacity, although the comb stabilizer comprises only ˜1wt % of the total polymer film. This observation is further evidencethat the combs remain localized to the film surface during coalescence.

Films coalesced from the RGD-bearing PMMA latex were prepared and seededwith WT NR6 cells as before. In contrast to latexes stabilized with thenon-cell binding combs, coalesced films of the RGD-bearing latexelicited cell attachment and spreading. Apparently, surface densities ofRGD ligand obtained for these latex films are comparable to the pureRGD-linked comb, although the latex film contains {fraction (1/100)} asmuch total peptide. Specificity of the adhesion of cells to theRGD-bearing surface was confirmed by adding excess soluble GRGDSP (45mM) to the culture media. All cells were observed to detach within 1hour of soluble RGD administration.

Modifications and variations of the present invention will be obvious tothose skilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe following claims.

We claim:
 1. A cell-regulating, polymeric composition comprising a blendof a cell-regulating, comb-type copolymer and one or more hydrophobic ornon-cell regulating polymers, wherein the cell-regulating, comb-typecopolymer comprises: a) a hydrophobic polymer backbone; b) non-cellhydrophilic polymeric side chains grafted to the polymer backbone,wherein the side chains have a molecular weight between 200 and 2000Daltons; wherein between zero and 100% of the non-cell binding,hydrophilic side chains are end-capped with cell-binding orcell-signaling ligands to form short cell-binding copolymer side chains;and wherein the side chains comprise less than 60% of the totalcopolymer weight, and wherein the blend contains between 1 and 99% byweight of the comb-type copolymer.
 2. The comb copolymer of claim 1having a total molecular weight of greater than 10,000 Daltons.
 3. Thepolymeric composition of claim 1, wherein the backbone is biodegradable.4. The polymeric composition of claim 1, wherein the backbone isnon-biodegradable.
 5. The polymeric composition of claim 1, wherein theside chains are less than 500 Daltons and constitute less than 60% ofthe total copolymer weight.
 6. The polymeric composition of claim 1wherein the mole percentage of backbone segments attached to hydrophilicside chains is between 2 and 30%.
 7. The polymeric composition of claim1 wherein the percent of hydrophilic side chains which includefunctional groups capable of being covalently or ionically attached to acell-binding or cell-signaling ligand is between 1 and 20%.
 8. Thepolymeric composition of claim 1, wherein the non-cell binding sidechains are selected from the group consisting of polyethylene glycol,polyethylene oxide polyacrylic acid and dextran.
 9. The polymericcomposition of claim 1, wherein the ligands are selected from the groupconsisting of adhesion peptides, cell-signaling peptides and growthfactors.
 10. The polymeric composition of claim 1 in a mixture furthercomprising non-cell-binding comb copolymers whose side chains are notend-capped with cell-binding or cell-signaling ligands.
 11. Thepolymeric composition of claim 10 wherein less than 20% of the combcopolymers comprise side chains that are end-capped with cell-binding orcell-signaling ligands.
 12. A tissue engineering matrix, cell culturematrix, biomedical device, or implant formed of or coated with a blendof a cell-regulating, comb-type copolymer and one or more hydrophobic ornon-cell regulating polymers, wherein the cell-regulating, comb-typecopolymer comprises: a) a hydrophobic polymer backbone; b) non-cellbinding hydrophilic polymeric side chains grafted to the polymerbackbone, wherein the side chains have a molecular weight between 200and 2000 Daltons; wherein between zero and 100% of the non-cell binding,hydrophilic side chains are end-capped with cell-binding orcell-signaling ligands to form short cell-binding copolymer side chains;wherein the side chains comprise less than 60% of the total copolymerweight, wherein the comb copolymer is effective in regulating cellularadhesion or response to the surface, and wherein the blend containsbetween 1 and 99% by weight of the comb-type copolymer.
 13. The tissueengineering matrix, cell culture matrix, biomedical device or implant ofclaim 12 seeded with cells selected from the group consisting ofparenchymal cells, skin cells, muscle cells, cartilage cells, nervecells and bone cells.
 14. The tissue engineering matrix, cell culturematrix, biomedical device or implant of claim 12 wherein thecell-regulating, comb-type copolymer comprises defined mixtures ofnon-cell binding and ligand-modified cell-regulating, comb-typecopolymers.
 15. The tissue engineering matrix, cell culture matrix,biomedical device or implant of claim 14, wherein the surface presentsdiscrete nanodomains or clusters of a single ligand type against abackground of non-cell binding hydrophilic side chains.
 16. The tissueengineering matrix, cell culture matrix, biomedical device or implant ofclaim 15, wherein each nanodomain or cluster contains between 2 and 50cell-signaling ligands in an area of 0.0001-0.01 microns square, withthe overall spacing between the edges of such domains in the range 3nm-200 nm.
 17. The tissue engineering matrix, cell culture matrix,biomedical device or implant of claim 14, wherein the surface presentsdiscrete nanodomains or clusters of two or more ligand types against abackground of non-cell binding hydrophilic side chains.
 18. The tissueengineering matrix, cell culture matrix, biomedical device or implant ofclaim 17, wherein each nanodomain or cluster contains between 2 and 50cell-signaling ligands in an area of 0.0001-0.01 microns square, withthe overall spacing between the edges of such domains in the range 3nm-200 nm.
 19. A method for making a tissue engineering matrix, cellculture matrix, implant or biomedical device with regulated cellularadhesion or response comprising coating or forming the matrix, implantor device with a blend of a cell-regulating, comb-type copolymer and oneor more hydrophobic or non-cell regulating polymers, wherein thecell-regulating, comb-type copolymer comprising: a) a hydrophobicpolymer backbone; b) non-cell binding hydrophilic polymeric side chainsgrafted to the polymer backbone, wherein between zero and 100% of thenon-cell binding, hydrophilic side chains are end-capped withcell-binding or cell-signaling ligands to form short cell-bindingcopolymer side chains; and wherein the side chains comprise less than60% of the total copolymer weight, and wherein the blend containsbetween 1 and 99% by weight of the comb-type copolymer.
 20. The methodof making a tissue engineering matrix, cell culture matrix, biomedicaldevice or implant of claim 19 in which non-cell binding side chains ofthe comb copolymers at the surface are end-capped with ligands after thecoating, matrix, device or implant is formed.
 21. A method forengineering tissue comprising growing cells on a tissue-engineeringmatrix formed of or coated with a blend of a cell-regulating, comb-typecopolymer and one or more hydrophobic or non-cell regulating polymers,wherein the cell-regulating, comb-type copolymer comprises: a) ahydrophobic polymer backbone; b) non-cell binding hydrophilic polymericside chains grafted to the polymer backbone, wherein between zero and100% of the non-cell binding, hydrophilic side chains are end-cappedwith cell-binding or cell-signaling ligands to form short cell-bindingcopolymer side chains; and wherein the side chains comprise less than60% of the total copolymer weight, wherein the comb copolymer iseffective in regulating cellular adhesion or response to the surface,and wherein the blend contains between 1 and 99% by weight of thecomb-type copolymer.